Our long-term goal is to understand the function of BRCT domain-containing proteins in cellular signaling. The BRCT motif was first identified as a tandem pair in the breast cancer protein BRCA1, and later in other proteins involved in DNA repair and cell cycle control. We showed that the BRCT domain is a phosphoprotein binding motif. While BRCA1 utilizes a BRCT tandem repeat, other proteins, including topoisomerase II binding protein, TopBP1, and DNA ligase IIIalpha, recognize their phosphorylated targets by means of a homodimer of BRCT domains. There is also strong evidence that NBS1 (Nijmegen Breakage Syndrome) protein binds phosphorylated histone H2AX through a tandem of BRCT and FHA domains. These observations that BRCT proteins bind phosphorylated targets through differently coupled BRCT domains - i.e., BRCT tandem repeats, BRCT homodimer, BRCT-FHA tandem domains - indicate that at least 3 modes of phosphoprotein recognition exist. There is no 3D structure of a BRCT/phosphoprotein complex. To understand how phosphoprotein recognition is achieved, we will use NMR spectroscopy, microcalorimetry and biochemistry techniques to determine the 3D structures, and probe the energetics of interaction of protein-phosphopeptide complexes illustrating all 3 BRCT binding modes. We will also test the hypothesis that phosphopeptide binding modulates other BRCT-mediated interactions.
Our specific aims are:
Aim 1. Probe the interaction of BRCA1 with phosphorylated peptides from the helicase BACH1 and CtIP corepressor to elucidate the molecular mechanism of phosphoprotein recognition by a BRCT tandem repeat.
Aim 2. Probe the interaction between TopBP1 and a phosphorylated peptide from E2F1 transcription factor to elucidate the molecular mechanism of phosphoprotein recognition by a BRCT homodimer.
Aim 3. Probe the interaction of NBS1 with a phosphorylated histone H2AX peptide to elucidate the molecular mechanism of phosphoprotein recognition by FHA-BRCT tandem domains.
Aim 4. Investigate a possible regulatory effect of phosphoprotein binding to DNA ligase IIIa on its BRCT-mediated interaction with DNA repair protein XRCC1.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Biophysical Chemistry Study Section (BBCB)
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Knowlton, John R
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Mayo Clinic, Rochester
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